Modified anti-cd52 antibody

ABSTRACT

The present invention provides for modified forms of anti-CD52 antibodies with reduced numbers of potential T-cell epitopes that are expected to display decreased immunogenicity.

This application claims priority to U.S. provisional application60/516,210, filed Nov. 1, 2003, the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to polypeptides to be administered,especially to humans and in particular for therapeutic use. Thepolypeptides are modified polypeptides, whereby the modification resultsin a reduced number of potential T-cell epitopes that provides a reducedpropensity for the polypeptide to elicit an immune response uponadministration to a human subject. The invention in particular relatesto the modification of antibodies reactive to the CD52 human leukocyteantigen to provide anti-CD52 antibodies that have a reduced number ofpotential T-cell epitopes, but retain the ability to bind to CD52.

BACKGROUND OF THE INVENTION

There are many instances whereby the efficacy of a therapeutic proteinis limited by an unwanted immune reaction to the therapeutic protein.Several mouse monoclonal antibodies have shown promise as therapies in anumber of human disease settings but in certain cases have failed due tothe induction of significant degrees of a human anti-murine antibody(HAMA) response [Schroff et al. (1985) Cancer Res. 45: 879-885; Shawleret al. (1985) J. Immunol. 135: 1530-1535]. For monoclonal antibodies, anumber of techniques have been developed in attempt to reduce the HAMAresponse [WOA8909622; EPA0239400; EPA0438310; WOA9106667; EPA0699755].These recombinant DNA approaches have generally reduced the mousegenetic information in the final antibody construct whilst increasingthe human genetic information in the final construct. Notwithstanding,the resultant “humanised” antibodies have, in several cases, stillelicited an immune response in patients [Issacs J. D. (1990) Sem.Immunol. 2: 449, 456; Rebello et al (1999) Transplantation 68:1417-1420].

Antibodies are not the only class of polypeptide molecule administeredas a therapeutic agent against which an immune response may be mounted.Even proteins of human origin and with the same amino acid sequences asoccur within humans can still induce an immune response in humans.Notable examples include therapeutic use of granulocyte-macrophagecolony stimulating factor [Wadhwa et al., (1999) Clin. Cancer Res. 5:1353-1361] and interferon α2 [Russo et al. (1996) Bri. J. Haem. 94:300-305; Stein et al. (1988) New Engl. J. Med. 318: 1409-1413] amongstothers.

Key to the induction of an immune response is the presence within theprotein of peptides that can stimulate the activity of T-cells viapresentation on MHC class II molecules, so-called “T-cell epitopes.”Such T-cell epitopes are commonly defined as any amino acid residuesequence with the ability to bind to MHC Class II molecules. Implicitly,a “T-cell epitope” means an epitope which when bound to MHC moleculescan be recognized by a T-cell receptor (TCR), and which can, at least inprinciple, cause the activation of these T-cells by engaging a TCR topromote a T-cell response.

MHC Class II molecules are a group of highly polymorphic proteins whichplay a central role in helper T-cell selection and activation. The humanleukocyte antigen group DR (HLA-DR) are the predominant isotype of thisgroup of proteins however, isotypes HLA-DQ and HLA-DP perform similarfunctions. In the human population, individuals bear two to four DRalleles, two DQ and two DP alleles. The structure of a number of DRmolecules has been solved and these appear as an open-ended peptidebinding groove with a number of hydrophobic pockets which engagehydrophobic residues (pocket residues) of the peptide [Brown et al.,Nature (1993) 364: 33; Stern et al. (1994) Nature 368: 215].Polymorphism identifying the different allotypes of class II moleculecontributes to a wide diversity of different binding surfaces forpeptides within the peptide binding grove and at the population levelensures maximal flexibility with regard to the ability to recogniseforeign proteins and mount an immune response to pathogenic organisms.

An immune response to a therapeutic protein proceeds via the MHC classII peptide presentation pathway. Here exogenous proteins are engulfedand processed for presentation in association with MHC class IImolecules of the DR, DQ or DP type. MHC Class II molecules are expressedby professional antigen presenting cells (APCs), such as macrophages anddendritic cells amongst others. Engagement of a MHC class II peptidecomplex by a cognate T-cell receptor on the surface of the T-cell,together with the cross-binding of certain other co-receptors such asthe CD4 molecule, can induce an activated state within the T-cell.Activation leads to the release of cytokines further activating otherlymphocytes such as B cells to produce antibodies or activating T killercells as a full cellular immune response.

T-cell epitope identification is the first step to epitope eliminationas recognised in WO98/52976 and WO00/34317. In these teachings,predicted T-cell epitopes are removed by the use of judicious amino acidsubstitutions within the protein of interest. Besides computationaltechniques, there are in vitro methods for measuring the ability ofsynthetic peptides to bind MHC class II molecules. An exemplary methoduses B-cell lines of defined MHC allotype as a source of MHC class IIbinding surface and may be applied to MHC class II ligand identification[Marshall et al. (1994) J. Immunol. 152:4946-4956; O'Sullivan et al.(1990) J. Immunol. 145: 1799-1808; Robadey et al. (1997) J. Immunol 159:3238-3246]. However, such techniques are not adapted for the screeningmultiple potential epitopes to a wide diversity of MHC allotypes, norcan they confirm the ability of a binding peptide to function as aT-cell epitope.

Recently, techniques exploiting soluble complexes of recombinant MHCmolecules in combination with synthetic peptides have come into use[Kern et al. (1998) Nature Medicine 4:975-978; Kwok et al (2001) TRENDSin Immunol. 22:583-588]. These reagents and procedures are used toidentify the presence of T-cell clones from peripheral blood samplesfrom human or experimental animal subjects that are able to bindparticular MHC-peptide complexes and are not adapted for the screeningmultiple potential epitopes to a wide diversity of MHC allotypes.

As depicted above and as consequence thereof, it would be desirable toidentify and to remove or at least to reduce potential T-cell epitopesfrom a given in principal therapeutically valuable but originallyimmunogenic peptide, polypeptide or protein. One of thesetherapeutically valuable molecules is a monoclonal antibody with bindingspecificity for the CD52 human leukocyte antigen. The preferredmonoclonal antibody of the present invention is a modified form of therat antibody termed “CAMPATH”. It is an objective of the invention toprovide for modified forms of the CAMPATH antibody with one or morepotential T-cell epitopes removed.

The CD52 molecule has a molecule weight of 21-28 kDa, and the matureprotein comprises a 12 amino acid peptide with a N-linkedoligosaccharide being attached to the membrane by itsglycophosphatidylinositol anchor. The antigen is present on at least 95%of human peripheral blood lymphocytes and also cells of themonocyte/macrophage series and in addition spermatozoa. It is notpresent on erythrocytes, platelets, tissue dendritic cells or bonemarrow stem cells (Hale et al. (1990) Tissue Antigens 35:873; Buggins etal (2002) Blood, 100:1715).

The first CD52 antibodies were raised in a rat immunized with humanlymphocytes in an attempt to obtain antibodies that activated complementfor use to deplete donor marrow of T-cells prior to transplantation[Hale et al. (1983) Blood 62: 873-882]. The majority of lytic antibodieswere anti-CD52 IgM antibodies. Although useful ex vivo, CD52 IgM(CAMPATH-1M) mediated complement activation was not effective ineliminating T-cells in vivo. CAMPATH-1G, a rat IgG2b monoclonalantibody, obtained by isotype switching from an IgG2a antibody clone,binds human Fc receptors, mediates cell death antibody-mediated cellulartoxicity (ADCCD) and is effective in eliminating cells in vivo [Friendet al (1991) Transplant. Proc. 23: 2253-2254; Hale et al (1998) Blood92: 4581-4590]. However, use of CAMPATH-1G is limited by the immuneresponse elicited in patients [Cobbold, J. S. (1990) J. Immunol. Methods127: 19-24; Dyer, M. J. S. (1989) Blood 73: 1431-1439]. To reduceimmunogenicity, a humanized IgG1 antibody, CAMPATH-1H, was engineered bycloning the Kabat hypervariable regions into a framework provided fromhuman NEW and Rei myeloma proteins [Riechmann et al., (1988) Nature 332:323-327]. Although reducing the immunogenicity compared to CAMPTH-1G,the humanized antibody still elicits immune responses in some patients.In an early report of treatment for rheumatoid arthritis, noantiglobulin response was reported in the 8 patients treated by a firstcourse of i.v. administration, but 3 of 4 patients who received a secondcourse of CAMPATH-1H developed antiglobulin antibodies (Issacs et al.(1992) Lancet, 21:1103-06). In a subsequent single-dose escalation i.v.study in rheumatoid arthritis patients, 63% of subjects developedantiglobulin responses, which were primarily anti-idiotypic responses[Weinblatt et al. (1995) Arthritis. Rheum. 38: 1589-1594]. Antiglobulinresponses were also reported for all 10 rheumatoid arthritis patientswho received escalating doses of CAMPATH-1H by subcutaneousadministration (Schnitzer et al., J. Rheumatol. (1997) 24:1031-36).

Thus, it is desirable to provide anti-CD52 antibodies with a reducednumber of potential T-cell epitopes which may result in a reduced orabsent potential to induce an immune response in the human subject. Suchproteins may be expected to display an increased circulation time withina human subject capable of mounting an immune response to thenon-modified antibody and may be of particular benefit in chronic orrecurring disease settings such as is the case for a number ofindications for CAMPATH. The present invention accordingly provides formodified forms of an anti-CD52 antibody with reduced numbers ofpotential T-cell epitopes that are expected to display decreasedimmunogenicity while however, substantially retaining the beneficialtherapeutic features associated with the efficacy of the parentalnon-modified antibody.

The invention discloses sequences identified within the variable regionsequences of the heavy and light chains of an anti-CD52 antibody thatare potential T cell epitopes by virtue of MHC class II bindingpotential.

While others have provided anti-CD52 antibody molecules including“humanised” forms [U.S. Pat. Nos. 5,846,543; 6,120,766; 6,569,430;WO0230460] none of these teachings recognise the importance of T cellepitopes to the immunogenic properties of the protein nor have beenconceived to directly influence said properties in a specific andcontrolled way according to the scheme of the present invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 depicts an exemplified vector for a modified heavy chain, “campVH.” dhfr is dihydrofolate reductase selection; CMV pro is the CMV IEpromoter; pA is Poly A; and Amp R is ampicillin resistance.

FIG. 2 depicts an exemplified vector for a modified light chain, “campVL”. Neo is neomycin (G148) selection; CMV pro is the CMV IE promoter;pA is Poly A; and Amp R is ampicillin resistance

FIG. 3 depicts the DNA and amino acid sequences of modified heavy chainvariable region DIVHv1.

FIG. 4 depicts the DNA and amino acid sequences of modified heavy chainvariable region DIVHv2.

FIG. 5 depicts the DNA and amino acid sequences of modified heavy chainvariable region DIVHv3.

FIG. 6 depicts the DNA and amino acid sequences of modified heavy chainvariable region DIVHv4.

FIG. 7 depicts the DNA and amino acid sequences of modified heavy chainvariable region DIVHv5.

FIG. 8 depicts the DNA and amino acid sequences of modified light chainvariable region DIVKv1.

FIG. 9 depicts the DNA and amino acid sequences of modified light chainvariable region DIVKv2.

FIG. 10 depicts the DNA and amino acid sequences of modified light chainvariable region DIVKv3.

FIG. 11 depicts the DNA and amino acid sequences of modified light chainvariable region DIVKv4.

FIG. 12 depicts the DNA and amino acid sequences of modified light chainvariable region DIVKv5.

FIG. 13 depicts the DNA and amino acid sequence of human IgG1 constantregion.

FIG. 14 depicts the DNA and amino acid sequence of human kappa constantregion.

FIG. 15 summarizes the results of the preliminary studies using thealternative dendritic cell:T cell protocol with the modifiedDIVHv5/DIVKv2 antibody.

FIG. 16 summarizes the comparison of T cell immunogenicity (dendriticcell:T cell assay) of humanised CAMPATH 1H and the modifiedDIVHv5/DIVKv2 antibody. Cpm values were compared (*) against untreatedcontrols using Students T-Test (p<0.05)

SUMMARY AND DESCRIPTION OF THE INVENTION

The present invention provides for a modified antibody in which theimmune characteristic is modified by means of reduced numbers ofpotential T-cell epitopes. Disclosed are sequences identified within theCAMPATH-1G variable region sequences of both the heavy chain and lightchain that are potential T-cell epitopes by virtue of MHC class IIbinding potential. The invention discloses the major regions of theantibody V-region sequence that may be immunogenic in man and modifiedsequences to eliminate or reduce the potential immunogenic effectivenessof these sites.

In one aspect, the invention provides a modified antibody moleculehaving specificity for the CD52 antigen recognised by the rat antibodyCAMPATH-1G wherein one or more amino acids in the variable region of theCAMPATH-1G antibody is substituted to reduce MHC class II recognition ofpeptides derived from this region. Implicit in the terms “anti-CD52antibody” and “CAMPATH antibody,” when applied to modified antibodies ofthe present invention, is an ability for such modified antibodies toretain an ability to bind to CD52. Embodiments of the inventionencompass an anti-CD52 antibody comprising a heavy chain V-regioncomprising a substituted variant of SEQ ID NO: 1 with one or more of thesubstitutions listed in Table 1, wherein the numbering of amino acidresidues relates to those of SEQ ID NO: 1, and comprising a light chainV-region comprising a substituted variant of SEQ ID NO: 2 with one ormore of the substitutions listed in Table 2, wherein the numbering ofamino acid residues relates to those of SEQ ID NO: 1. In someembodiments the anti-CD52 antibody heavy chain further comprise a humanIgG1 constant region domain and the light chain further comprises ahuman kappa constant region domain.

TABLE 1 Substitutions within potential T-cell epitopes in the CAMPATH-1Gvariable heavy chain (SEQ ID NO: 1) VH WT Residue # residue Substitution3 K Q 5 L A C B Z G H K P R S T 12 V B E H K P Q R S T 13 Q A F H K N PQ R S T 15 G D H P Q R S T 17 S G M P W 18 M A G P L 19 R A C F G I L MP V W Y 20 L A C F G H I K B M Z P R S T V W Y 21 S P 23 A B Z G H K P RS T 25 S F G L P W Y 26 G B Z H K P R S T W Y 31 D A F G I M P V W Y 33Y A G M P 35 N P 36 W A D E G H K N P Q R S T 37 I V 38 R F H P Y 40 P A41 A B Z H K P R S T W 42 G I P T Y 44 A G H N P Q S T W Y 45 P L 48 L VI 71 T F L P W Y 72 I D E H K N P Q R S T 73 S A G P 74 R A F G I M P WY 76 N A G M P W Y 77 T A H I P S 78 Q K 79 N A F G I M P V W Y 80 M A DE G H K N P Q R T S 82 Y A D E G H K N P Q R S T 84 Q A F G I L M P V WY 85 M A D E G H K N P Q R S T 87 T S 88 L D E G H K N P Q R S T 89 R FP W Y 90 A B Z H K P R S T W Y 91 E P 92 D A F G I L M P V W Y 95 T V109 D A F G I L M P V W Y 111 W A D E G H K N P Q R S T 114 G H P S T115 V T 116 M L F I P T V W Y 117 V A F G I M P W Y

TABLE 2 Substitutions within potential T-cell epitopes in the CAMPATH-1Glight chain (SEQ ID NO: 2) VK WT Residue # residue Substitution 3 K Q 10F A B Z G H K P R S T 15 V A G H P 17 D P 19 V P W 21 L P I 22 N T 24 KR 33 L A B Z G H K P R S T 40 L B Z G H K P R S T 42 E K 43 S A 46 L S56 T A F G I M P S W Y 58 I A G M P V 60 S A F G I M P W Y 61 R P 63 S FL P W Y 64 G B Z H K P R S T 78 L B Z G H K P R S T 83 V A B Z G H I K PR S T 87 F Y

In various embodiments, more than 2 amino acid substitutions, or morethan 3 amino acid substitutions, or more than 4 amino acidsubstitutions, or more than 5 amino acid substitutions, or more than 6amino acid substitutions, or more than 7 amino acid, or more than 8, ormore than 9, or more than 10, or more than 11 or more than 12substitutions are made in the heavy chain and/or the light chain. Insome embodiments, between 5 and 20, or between 7 and 14, amino acidsubstitutions are made in the heavy and/or light chain.

In some embodiments, the anti-CD52 antibody comprises a V-region heavychain comprising a substituted variant of SEQ ID NO: 1 with one or moreof the following substitutions, wherein the numbering of amino acidresidues relates to those of SEQ ID NO: 1:

-   -   substitution of Lys at amino acid residue 3 with Gln;    -   Leu at amino acid residue 5 with Ala, Cys, Asn, Asp, Gln, Glu,        Gly, His, Lys, Pro, Arg, Ser, or Thr;    -   Met at amino acid residue 18 with Arg, Gly, Pro, Leu;    -   Leu at amino acid residue 20 with Ala, Cys, Phe, Gly, His, Ile,        Lys, Asn, Asp, Met, Gln, Glu, Pro, Arg, Ser, Thr, Val Trp, or        Tyr;    -   Ala at amino acid residue 23 with Asp, Asn, Glu, Gln, Gly, His,        Lys, Pro, Arg, Ser, Thr;    -   Ile at amino acid residue 37 with Val;    -   Pro at amino acid residue 40 with Ala;    -   Ala at amino acid residue 41 with Pro;    -   Ala at amino acid residue 44 with Gly, His, Asn, Pro, Gln, Ser,        Thr, Trp, Tyr;    -   Pro at amino acid residue 45 with Leu;    -   Leu at amino acid residue 48 with Ile or Val;    -   Thr at amino acid residue 77 with Ala, His, Ile, Pro or Ser;    -   Gln at amino acid residue 78 with Lys;    -   Met at amino acid residue 80 with Ala, Asp, Glu, Gly, His, Lys,        Asn, Pro, Gln, Arg, Thr, or Ser;    -   Tyr at amino acid residue 82 with Ala, Asp, Glu, Gly, His, Lys,        Asn, Pro, Gln, Arg, Ser or Thr;    -   Met at amino acid residue 85 with Ala, Asp, Glu, Gly, His, Lys,        Asn, Pro, Gln, Arg, Ser or Thr;    -   Thr at amino acid residue 87 with Ser;    -   Thr at amino acid residue 95 with Val;    -   Val at amino acid residue 115 with Thr;    -   Met at amino acid residue 116 with Thr, Phe, Ile, Leu, Pro, Val,        Trp or Tyr;    -   and comprising a V-region light chain comprising a substituted        variant of SEQ ID NO: 2 with one or more of the following        substitutions, wherein the numbering of amino acid residues        relates to those of SEQ ID NO: 2:    -   substitution of Lys at amino acid residue 3 with Gln Phe at        amino acid residue 10 with Ala, Asp, Asn, Glu, Gln, Gly, His,        Lys, Pro, Arg, Ser or Thr;    -   Leu at amino acid residue 21 with Pro or Ile;    -   Asn at amino acid residue 22 with Thr;    -   Lys at amino acid residue 24 with Arg;    -   Leu at amino acid residue 40 with Asp, Asn, Gln, Glu, Gly, His,        Lys, Pro, Arg, Ser or Thr;    -   Glu at amino acid residue 42 with Lys;    -   Ser at amino acid residue 43 with Ala;    -   Leu at amino acid residue 46 with Ser;    -   Thr at amino acid residue 56 with Ala, Phe, Gly, Ile, Met, Pro,        Ser, Trp or Tyr;    -   Ile at amino acid residue 58 with Ala; Gly, Met, Pro or Val;    -   Val at amino acid residue 83 with Ala, Asp, Asn, Glu, Gln, Gly,        His, Ile, Lys, Pro, Arg, Ser, Thr; and    -   Phe at amino acid residue 87 with Tyr.

In some embodiments of the present invention, the anti-CD52 antibodycomprises a V-region heavy chain comprising a substituted variant of SEQID NO: 1 with one or more of the following substitutions, wherein thenumbering of amino acid residues relates to those of SEQ ID NO: 1:

-   -   substitution of Lys at amino acid residue 3 with Gln;    -   Leu at amino acid residue 5 with Gln;    -   Met at amino acid residue 18 with Leu;    -   Leu at amino acid residue 20 with Ile;    -   Ala at amino acid residue 23 with Ser;    -   Ile at amino acid residue 37 with Val;    -   Pro at amino acid residue 40 with Ala;    -   Ala at amino acid residue 41 with Pro;    -   Ala at amino acid residue 44 with Gly;    -   Pro at amino acid residue 45 with Leu;    -   Leu at amino acid residue 48 with Ile or Val;    -   Thr at position 77 with Ala or Ser;    -   Gln at amino acid residue 78 with Lys;    -   Met at amino acid position 80 with Thr, or Ser;    -   Tyr at amino acid residue 82 with His;    -   Met at amino acid residue 85 with Ala;    -   Thr at amino acid residue 87 with Ser;    -   Thr at amino acid residue 95 with Val;    -   Val at amino acid residue 115 with Thr;    -   Met at amino acid residue 116 with Leu;    -   and comprising a V-region light chain comprising a substituted        variant of SEQ ID NO: 2 with one or more of the following        substitutions, wherein the numbering of amino acid residues        relates to those of SEQ ID NO: 2:    -   substitution of Lys at amino acid residue 3 with Gin;    -   Phe at amino acid residue 10 with Ser;    -   Leu at amino acid residue 21 with Ile;    -   Asn at amino acid residue 22 with Thr;    -   Lys at amino acid residue 24 with Arg;    -   Leu at amino acid residue 40 with Pro;    -   Glu at amino acid residue 42 with Lys;    -   Ser at amino acid residue 43 with Ala;    -   Leu at amino acid residue 46 with Ser;    -   Thr at amino acid residue 56 with Ser;    -   Ile at amino acid residue 58 with Val;    -   Val at amino acid residue 83 with Ile;    -   Phe at amino acid residue 87 with Tyr.

In a further aspect of the invention, there are provided variantmonoclonal antibodies with a reduced number of potential T-cellepitopes, said variants comprising a combination of heavy chain V-regioncomprising a sequence selected from SEQ ID NO: 3 through SEQ ID NO: 7 orSEQ ID NO: 13 through SEQ ID NO: 40 and light chain V-regions comprisinga sequence selected from SEQ ID NO: 8 through SEQ ID NO: 12 or SEQ IDNO: 41 through SEQ ID NO: 70. In some preferred embodiments, theinvention provides for variant monoclonal antibodies with a reducednumber of potential T-cell epitopes, said variants comprising acombination of heavy chain V-region comprising a sequence selected fromSEQ ID NO: 3 through SEQ ID NO:7 and light chain V-region comprising asequence selected from SEQ ID NO: 8 through SEQ ID NO: 12. In someembodiments the anti-CD52 antibody further comprises a human IgG1constant region domain and a human kappa constant region domain. Infurther embodiments, the anti-CD52 antibody comprising a human IgG1constant region and a human kappa constant region comprises a heavychain V-region comprising SEQ ID NO: 4 and a light chain V-regioncomprising SEQ ID NO: 12, or a heavy chain V-region comprising SEQ IDNO: 7 and a light chain V-region comprising SEQ ID NO: 12, or a heavychain V-region comprising SEQ ID NO: 7 and a light chain V-regioncomprising SEQ ID NO: 10, or a heavy chain V-region comprising SEQ IDNO: 3 and a light chain V-region comprising SEQ ID NO: 10, or a heavychain V-region comprising SEQ ID NO: 6 and a light chain V-regioncomprising SEQ ID NO: 10.

The present invention also encompasses an accordingly specifiedmolecule, wherein the alteration of the amino acid residues issubstitution, addition or deletion of originally present amino acid(s)residue(s) by other amino acid residue(s) at specific position(s); anaccordingly specified molecule, wherein, if necessary, additionallyfurther alteration usually by substitution, addition or deletion ofspecific amino acid(s) is conducted to restore a biological activity ofsaid molecule; an accordingly specified molecule wherein alteration isconducted at one or more residues from any or all of the string ofcontiguous residues of sequences (A)-(S) as below wherein said sequencesare derived from the CAMPATH-1G antibody V-region sequence domains ofthe molecule and where using single letter code;

A. = KLLESGGGLVQPG; B. = GLVQPGGSMRLSC; C. = GSMRLSCAGSGFT; D.= DFYMNWIRQPAGK; F. = MNWIRQPAGKAPE; F. = FTISRDNTQNMLY; G.= QNMLYLQMNTLRA; H. = MLYLQMNTLRAED; I. = LQMNTLRAEDTAT; J.= NTLRAEDTATYYC; K. = DYWGQGVMVTVSS; L. = PSFLSASVGDRVT; M.= ASVGDRVTLNCKA; N. = DRVTLNCKASQNI; O. = KYLNWYQQKLGES; P.= QKLGESPKLLIYN; Q. = TGIPSRFSGSGSG; R. = SSLQPEDVATYFC; S.= EDVATYFCLQHIS.

One aspect of the present invention is a pharmaceutical compositioncomprising a modified CAMPATH-1G molecule modified so as to reduce thenumber of potential T-cell epitopes and having the ability to bind toCD52, wherein said composition comprises a pharmaceutically acceptablecarrier.

Another aspect of the present invention is an expression vectorcomprising a nucleic acid sequence coding a modified heavy or lightchain of the present invention. In some embodiments, the expressionvector comprises a nucleic acid sequence encoding a V-region heavy chaincomprising a modified substituted variant of SEQ ID NO: 1 with a reducednumber of potential T-cell epitopes, operably linked to an expressioncontrol sequence. In various embodiments, the expression vectorcomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 71 through SEQ ID NO: 75 and SEQ ID NO: 81 through SEQ ID NO:108, or a degenerate variant thereof. Degeneracy in relation topolynucleotides refers to the fact well recognized in the art that inthe genetic code many amino acids are specified by more than one codon.The degeneracy of the code accounts for 20 different amino acids encodedby 64 possible triplet sequences of the four different bases. In someembodiments, the expression vector comprises a nucleic acid sequenceencoding a V-region light chain comprising a modified substitutedvariant of SEQ ID NO: 2 with a reduced number of potential T-cellepitopes, operably linked to an expression control sequence. In variousembodiments, the expression vector comprises a nucleic acid sequenceselected from the group consisting of SEQ ID NO: 76 through SEQ ID NO:80 and SEQ ID NO: 109 through SEQ ID NO: 138, or degenerate variantthereof. An example of a suitable expression vector for a heavy chain ofthe present invention is shown in FIG. 1 and an example of a suitableexpression vector for a light chain of the present invention is shownFIG. 2. Another aspect of the present invention is a cultured cellcomprising one or more of the aforementioned vectors. A further aspectof the present invention is a method of preparing an immunoglobulin,comprising culturing the aforementioned cell under conditions permittingexpression under the control of suitable expression control sequence(s),and purifying the immunoglobulin from the medium of the cell.

Other aspects of the present invention are methods of therapeutictreatment. Embodiments encompass a method of treating lymphoidmalignancies comprising administering to a patient an effective amountof a modified antibody according to the present invention. In someembodiments, the lymphoid malignancy is leukemia or lymphoma. Otherembodiments include a method of treating autoimmune conditions in apatient comprising administering an effective amount of a modifiedantibody according to the present invention. In various embodiments theautoimmune condition is multiple sclerosis, rheumatoid arthritis,systemic vasiculitis, uveitis, inflammatory bowel disease orscleroderma.

Embodiments also include a method of immunosuppressing a patient priorto or subsequent to transplantation of an organ comprising administeringto said patient an effective amount of an antibody according to thepresent invention. In some embodiments, the transplantation of on organis a renal transplant.

Reference to “substantially non-immunogenic” or “reduced immunogenicpotential” includes reduced immunogenicity compared to a parentantibody, i.e., a non-modified rodent or chimeric (rodent V-regions;human constant regions) monoclonal antibody or the humanized monoclonalantibody CAMPATH-1H. The term “immunogenicity” includes an ability toprovoke, induce or otherwise facilitate a humoral and or T-cell mediatedresponse in a host animal and in particular where the “host animal” is ahuman or the ability to elicit a response in a suitable in vitro assay,e.g., the dendritic cell/T-cell assay described herein.

A preferred feature of the modified antibodies of the present is thatthey substantially retain the functional activities of the non-modifiedor “parental” antibody CAMPATH-1G or the humanized antibody CAMAPATH-1H.Embodiments of the invention therefore encompass modified antibodies inwhich one or more of the beneficial technical features associated withthe therapeutic efficacy of CAMPATH-1H or the parental non-modifiedantibody are exhibited. Such modified antibodies are useful in a numberof important diseases in man including especially lymphoid malignanciessuch as leukemia and lymphoma, autoimmune conditions including, but notlimited to, multiple sclerosis, rheumatoid arthritis, systemicvasiculitis, uveitis, inflammatory bowel disease and scleroderma andalso for use in transplantations.

Accordingly, the modified antibody of the present exhibits an ability tobind to CD52 and in preferred embodiments the affinity for its targetantigen CD52 is within an order of magnitude higher or lower than theaffinity exhibited by the monoclonal antibody CAMPATH-1H.

The therapeutic efficacy of the parental molecule is believed also to bemediated by the ability of the antibody to induce antibody-dependentcell mediated cytotoxicity (ADCC) and complement dependent cytotoxicity(CDC). The phenomena of ADCC and CDC are mediated by the heavy chainconstant region domain of whole antibody molecules, and the presentinvention contemplates production of a whole antibody moleculescomprising a constant region domain compatible with ADCC and CDCinduction. In preferred embodiments, the modified antibody comprises ahuman IgG1 constant region and a human kappa constant region domain.

By “antibody” is meant a protein of the immunoglobulin family that iscapable of combining, interacting or otherwise associating with anantigen. The term “antigen” is used herein to refer to a substance thatis capable of interacting with the antibody and in the context of thepresent invention is meant to be CD52.

The term “immunoglobulin” is used herein to refer to a proteinconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes. The recognised immunoglobulin genes include the κ,λ, α, γ (IgG1, IgG2, IgG3, IgG4), σ, ε and μ constant region genes andin nature multiple immunoglobulin variable region genes. One naturalform of immunoglobulin is a tetramer comprising two identical pairs inwhich each pair has one light chain and one heavy chain. In each pairthe heavy and light chain variable regions together provide the bindingsurface capable of interacting with the antigen. The term Vh is usedherein to refer to the heavy chain variable region, and the term Vk isused herein to refer to the light chain variable region and in thisinstance in common with numerous monoclonal antibodies the light chainis a “kappa” (k) type chain.

As used herein, Vh means a polypeptide that is about 110 to 125 aminoacid residues in length, the sequence of which corresponds to any of thespecified Vh chains herein which in combination with a Vk are capable ofbinding CD52 antigen. Similarly, Vk means a polypeptide that is about95-130 amino acid residues in length the sequence of which correspondsto any of the specified Vk chains herein which in combination with a Vhare capable of binding the CD52 antigen. Full-length immunoglobulinheavy chains are about 50 kDa molecular weight and are encoded by a Vhgene at the N-terminus and one of the constant region genes (e.g., γ) atthe C-terminus. Similarly, full-length light chains are about 25 kDamolecular weight and are encoded by a V-region gene at the N-terminusand a κ or λ constant region gene at the C-terminus.

In addition to whole antibody (tetramers), immunoglobulins may exist ina number of other forms derived by application of recombinant DNAtechniques or protein biochemistry. These forms include for example Fv,Fab, Fab′ and (Fab)2 molecules and could all contain any of the Vh or Vksequences of the present invention. A further example may include a“bi-specific” antibody comprising a Vh/Vk combination of the presentinvention in combination with a second Vh/Vk combination with adifferent antigen specificity.

The term “potential T-cell epitope” means according to the understandingof this invention an amino acid sequence which has potential to bind MHCclass II. Such sequences may stimulate T-cells and/or bind (withoutnecessarily measurably activating) T-cells in complex with MHC class II.

The term “peptide” as used herein and in the appended claims, is acompound that includes two or more amino acids. The amino acids arelinked together by a peptide bond (defined herein below). There are 20different naturally occurring amino acids involved in the biologicalproduction of peptides, and any number of them may be linked in anyorder to form a peptide chain or ring. The naturally occurring aminoacids employed in the biological production of peptides all have theL-configuration. Synthetic peptides can be prepared employingconventional synthetic methods, utilizing L-amino acids, D-amino acids,or various combinations of amino acids of the two differentconfigurations. Some peptides contain only a few amino acid units. Shortpeptides, e.g., having less than ten amino acid units, are sometimesreferred to as “oligopeptides”. Other peptides contain a large number ofamino acid residues, e.g., up to 100 or more, and are referred to as“polypeptides”. By convention, a “polypeptide” may be considered as anypeptide chain containing three or more amino acids, whereas a“oligopeptide” is usually considered as a particular type of “short”polypeptide. Thus, as used herein, it is understood that any referenceto a “polypeptide” also includes an oligopeptide. Further, any referenceto a “peptide” includes polypeptides, oligopeptides, and proteins. Eachdifferent arrangement of amino acids forms different polypeptides orproteins. The number of polypeptides—and hence the number of differentproteins-that can be formed is practically unlimited.

The general method of the present invention leading to the modifiedanti-CD52 antibody comprises the following steps:

-   -   (a) Determining the amino acid sequence of the polypeptide or        part thereof.    -   (b) Identifying one or more potential T cell epitopes within the        amino acid sequence of the protein by any method including        determination of the binding of the peptides to MHC molecules        using in vitro or in silico techniques or biological assays.    -   (c) Designing new sequence variants with one or more amino acids        within the identified potential T cell epitopes modified in such        a way to substantially reduce or eliminate binding of the        peptides to MHC molecules measured by in vitro or in silico        techniques or biological assays. Such sequence variants are        created in such a way to avoid creation of new potential T cell        epitopes by the sequence variations unless such new potential T        cell epitopes are, in turn, modified in such a way to        substantially reduce or eliminate binding of peptides to MHC        class II molecules.    -   (d) Constructing such sequence variants by recombinant DNA        techniques and testing said variants in order to identify one or        more variants with desirable properties.

The identification of potential T-cell epitopes according to step (b)can be carried out according to methods described previously in the art.Suitable methods are disclosed in WO 98/59244; WO 00/34317; U.S.Application 20030153043, all incorporated herein by reference.

In practice a number of variant anti-CD52 antibodies may be produced andtested for the desired immune and functional characteristic. It isparticularly important when conducting alterations to the proteinsequence that the contemplated changes do not introduce new immunogenicepitopes. This event is avoided in practice by re-testing thecontemplated sequence for the presence of epitopes and or of MHC classII ligands by any suitable means.

In various embodiments, the modified antibodies of the present inventionare generated by expression of different combinations of the Vh and Vkgenes specified herein. All such combinations of heavy and light chainare encompassed by the present invention.

The invention relates to an anti-CD52 antibody in which substitutions ofat least one amino acid residue have been made at positions within theV-regions of the molecule to result in the elimination of one or morepotential T-cell epitopes from the protein. It is most preferred toprovide modified antibody molecules in which amino acid modification(e.g., a substitution) is conducted within the most immunogenic regionsof the parent molecule. The various embodiments of the present inventioncomprise modified antibody molecules for which any of the MHC class IIligands are altered such as to eliminate binding or otherwise reduce thenumbers of MHC allotypes to which the peptide can bind. The inventorshave discovered and herein disclose, the immunogenic regions of theCAMPATH antibody molecule in man. It is understood that under certaincircumstances additional regions of sequence to those disclosed hereincan become immunogenic epitopes, for example in the event of infectionwith a pathogen expressing a protein or peptide with a similar sequenceto that of the present case.

MHC class II epitope removal has involved amino acid substitution tocreate modified variants depleted of potential T-cell epitopes. Theamino acid substitutions have been made at appropriate points within thepeptide sequence predicted to achieve substantial reduction orelimination of the activity of the undesired potential T cell epitope.Examples of particularly useful substitutions in this respect areprovided in Tables 1 and 2, wherein Table 1 relates to Vh regionsubstitutions and Table 2 relates to Vk region substitutions.

As will be clear to the person skilled in art, multiple alternative setsof substitutions could be arrived at which achieve the objective ofremoving un-desired epitopes. The resulting sequences would howeverremain broadly homologous with the specific compositions disclosedherein and therefore fall under the scope of the present invention. Itwould be typical to arrive at sequences that were around 70%, or around90%, or around 95%, or around 99% or more homologous with the presentspecified sequences over their least homologous region and yet remainoperationally equivalent. Such sequences would equally fall under thescope of the present.

It is understood that single amino acid substitutions within a givenpotential T cell epitope are the most preferred route by which theepitope may be eliminated. Combinations of substitution within a singleepitope may be contemplated and for example can be particularlyappropriate where individually defined epitopes are in overlap with eachother. Moreover, amino acid substitutions either singly within a givenepitope or in combination within a single epitope may be made atpositions not equating to the “pocket residues” with respect to the MHCclass II binding groove, but at any point within the peptide sequence.All such substitutions fall within the scope of the present.

In as far as this invention relates to modified anti-CD52 antibodies,compositions containing such modified antibodies or fragments ofmodified antibodies and related compositions should be considered withinthe scope of the invention. The invention therefore contemplates the useand generation of antibody fragments including for example Fv, Fab, Fab′and F(ab′)2 fragments. Such fragments may be prepared by standardmethods [for example; Coligan et al., Current Protocols in Immunology,John Wiley & Sons 1991-1997, incorporated herein by reference]. Thepresent invention also contemplates the various recombinant forms ofantibody derived molecular species well known in the art. Such speciesinclude stabilised Fv fragments including single chain Fv forms (e.g.,scFv) comprising a peptide linker joining the Vh and Vk domains, or anFv stabilised by interchain di-sulphide linkage (dsFv) and which containadditional cysteine residues engineered to facilitate the conjoining ofthe Vh and Vk domains. Equally, other compositions are familiar in theart and could include species referred to as “minibodies”; and singlevariable domain “dAbs.” Other species still may incorporate means forincreasing the valency of the modified antibody V-region domain, i.e.species having multiple antigen binding sites for example by theengineering of dimerisation domains (e.g., “leucine zippers”) or alsochemical modification strategies.

Under the scheme of the present there are provided a number of differentH-chain V-region and L-chain V-region sequences. The present disclosureprovides no limit to the possible combinations of H-chain and L-chainthat may be provided to constitute a complete antibody molecule.Constitution of the complete antibody molecule may be achieved byrecombinant DNA techniques and methods for purifying and manipulatingantibody molecules well known in the art. Necessary techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual,” second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Handbook of Experimental Immunology” (D. M. Weir & C. C.Blackwell, eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M.Miller & M. P. Calos, eds., 1987); “Current Protocols in MolecularBiology” (Ausubel et al., eds., 1987); “PCR: The Polymerase ChainReaction,” (Mullis et al., eds., 1994); “Current Protocols inImmunology” (Coligan et al., eds., 1991).

The preferred molecules of this invention can be prepared in any ofseveral ways but is most preferably conducted exploiting routinerecombinant methods. It is a relatively facile procedure to use theprotein sequences and information provided herein to deduce apolynucleotide (DNA) encoding any of the preferred antibody V-regions.This can be achieved for example using computer software tools such asthe DNAstar software suite [DNAstar Inc, Madison, Wis., USA] or similar.Any such DNA sequence with the capability of encoding the preferredpolypeptides of the present or significant homologues thereof, should beconsidered as embodiments of this invention.

As a general scheme any of the Vh or Vk chain genes can be made usinggene synthesis and cloned into a suitable expression vector. In turn theexpression vector is introduced into a host cell and cells selected andcultured. The antibody molecules are readily purified from the culturemedium and formulated into a preparation suitable for therapeuticadministration.

By way of a non-limiting example, one such scheme involves a genesynthesis process using panels of synthetic oligonucleotides. The genesare assembled using a ligase chain reaction (LCR) wherein theoligonucleotides featuring complementary ends are allowed to annealfollowed by amplification and fill-in using a polymerase chain reaction(PCR). The PCR is driven by addition of an increased concentration ofthe flanking oligonucleotides to act as primers. The PCR products areassembled into full-length antibody genes by further PCR from vectorscontaining 5′ and 3′ immunoglobulin gene flanking regions andsub-cloning into expression vectors for expression of whole antibody.The assembled Vh and Vk genes can serve as templates for mutagenesis andconstruction of multiple variant antibody sequences such as any of thosedisclosed herein. It is particularly convenient to use the strategy of“overlap extension PCR” as described by Higuchi et al. [1988, NucleicAcids Res. 16: 7351], although other methodologies and systems could bereadily applied.

Full-length immunoglobulin genes containing the variable regioncassettes are most conveniently assembled using overlapping PCR andsub-cloned into expression vectors containing the desired immunoglobulinconstant region domains. The expression vectors may be introduced into amammalian or other host cell for example using electroporationtechniques. The NSO cell line is a non-immunoglobulin producing mousemyeloma, obtained from the European Collection of Animal Cell Cultures(ECACC) and is particularly suitable example host cell line for thisprocedure. Cell lines secreting antibody are expanded and antibody canbe readily purified for example by use of protein A affinitychromatography [Harlow & Lane, ibid]. The concentration of the purifiedantibody can be determined using an enzyme linked immunosorbent assay(ELISA) detecting the human kappa constant region of the antibodies ofinterest.

In a further aspect the present invention relates to methods fortherapeutic treatment of humans using the modified antibodycompositions. For administration to an individual, any of the modifiedantibody compositions would be produced to be preferably at least 80%pure and free of pyrogens and other contaminants. It is furtherunderstood that the therapeutic compositions of the modified antibodyproteins may be used in conjunction with a pharmaceutically acceptableexcipient. The pharmaceutical compositions according to the presentinvention are prepared conventionally, comprising substances that arecustomarily used in pharmaceuticals, e.g., Remington's PharmaceuticalSciences, (Alfonso R. Gennaro, ed., 18th edition, 1990), includingexcipients, carriers, adjuvants, and buffers. The compositions can beadministered, e.g., parenterally, enterally, intramuscularly,subcutaneously, intravenously, or other routes useful to achieve aneffect. For example: anti-CD52 antibodies can be given intravenously(Cloes et al. (1999) Ann. Neurol., 46:296-304; Moreau et al. (1996)Multiple Sclerosis, 1:357-65; Moreau et al. (1994) Lancet, 344:298-301,all herein incorporated by reference) and subcutaneously (Schnitzer etal. (1997) J. Rheumatol., 24:1031-6; Bowen et al. (1997) Br. J.Hematol., 96:617-9, both herein incorporated by reference). Conventionalexcipients include pharmaceutically acceptable organic or inorganiccarrier substances suitable for parenteral, enteral, and other routes ofadministration that do not deleteriously react with the agents. Forparenteral application, particularly suitable are injectable sterilesolutions, preferably oil or aqueous solutions, as well as suspensions,emulsions or implants, including suppositories. Ampoules are convenientunit dosages. The pharmaceutical preparations can be sterilized and, ifdesired, mixed with stabilizers, wetting agents, emulsifiers, salts forinfluencing osmotic pressure, buffers, or other substances that do notreact deleteriously with the active compounds.

In the methods of the present invention, the actual dosage of theanti-CD52 antibodies of the present invention employed will depend on avariety of factors including the type and seventy of disorder beingtreated, and other treatment modality or modalities selected. Guidancefor dosage regimens is obtained from dosing of CAMPATH-1H known in theart.

EXPERIMENTAL EXAMPLES Example 1 Construction of Anti-CD52 Antibody V_(H)and V_(K) Genes

The sequence of the rat anti-CD52 antibody was derived from data baseentries RNIGHCC1G for the variable domain heavy chain (V_(H)), andRNIGKCC1G for the variable domain light chain (V_(K)). The sequenceswere modified slightly to remove internal HindIII and BamHI siteswithout altering the amino acid sequence. The V_(H) and V_(K) genes weremade by gene synthesis. Briefly, a panel of synthetic oligonucleotideswas designed and synthesised. The genes were assembled using a ligasechain reaction (LCR) wherein the oligonucleotides featuringcomplementary ends were allowed to anneal followed by amplification andfill-in using a polymerase chain reaction (PCR). The PCR was driven byaddition of an increased concentration of the flanking oligonucleotidesto act as primers. The PCR products were assembled into full-lengthantibody genes by further PCR from vectors containing 5′ and 3′immunoglobulin gene flanking regions and sub-cloning into expressionvectors for expression of whole antibody. The assembled V_(H) and V_(K)genes served as templates for mutagenesis and construction of multiplevariant antibody sequences in which potential T-cell epitopes had beenremoved.

For assembly of the V_(H) gene oligonucleotides VH1 to VH20 detailed inTable 3 were used. For assembly of the V_(K) gene oligonucleotides VK1to VK20 detailed in Table 4 were used. For both genes, the LCR wasconducted by mixing 20 μl of phosphorylated oligonucleotides with 1 μlPfu DNA ligase (Stratagene, Amsterdam, NL), 10 μl 10× reaction buffer(supplied with enzyme) and 69 μl water. The reaction mix was placed in athermal cycler for incubation at 95° C. for 2 minutes followed by 25cycles of 95° C. for 30 seconds, gradual cooling to 50° C., incubationat 50° C. for 30 seconds, and 55° C. for 20 minutes, followed by a finalincubation of 3 hours at 55° C. Analysis of a sample of the LCR using 1%agarose gel electrophoresis gave a smear with a faint band of correctsize just visible. The oligonucleotides in all cases were fromSigma-Genosys (Pampisford, UK) and were phosphorylated in vitro using T4DNA kinase (Promega, Southampton, UK) and the supplier's recommendedprotocol. Following LCR, 5 μL of the reaction was transferred to a PCRmix to amplify the assembled fragment. Oligonucleotides VH1 and VH20were used to drive the V_(H) reaction, with oligonucleotides VK1 andVK20 used to drive the V_(K) reaction. PCR was conducted in a totalvolume of 50 μl for 30 cycles using 1 μl Pfu DNA polymerase (Stratagene,Amsterdam, NL). Analysis of a sample of the PCR using 1% agarose gelelectrophoresis gave a band of 380 bp for V_(H) and 377 bp for V_(K).

Variable region cassettes were assembled using overlapping PCR. Briefly,DNAs derived from the vectors M13-VHPCR1 and M13-VKPCR1 [Orlandi et al.(1989), PNAS, 89: 3833-7] were used as templates to produce a furthertwo overlapping PCR fragments for each V_(H) and V_(K) chain including5′ flanking sequence encoding the leader signal peptide and 3′ flankingsequence including a splice site and intron sequences. The DNA fragmentsso produced for the V_(H) and V_(K) were combined in a PCR usingflanking primers required to obtain full-length DNA sequences. Theprimer pairs used in these “linking” reactions were oligonucleotidesVHVK5′CHO/VHVK3′SIG and VH19/VH12 for the V_(H) gene, whereas for theV_(K) gene, the oligonucleotides VHVK5′CHO/VHVK3′SIG and VK19/VK3′CHOwere used.

After purification using a Qiagen (Crawley, UK) PCR PREP kit the PCRproducts were cut with HindIII and BamHI (Promega, Southampton, UK) andrun on a 1% agarose gel. The desired bands were removed and purifiedusing a Qiagen (Crawley, UK) DNA extraction kit. The products werecloned into HindIII and BamHI cut pUC19 vector and the DNA sequenceconfirmed.

TABLE 3 Oligonucleotides for synthesis of V_(H) gene SEQ Name SequenceID NO: VH1 TCCACAGGTGTCCACTCCGA 141 VH2CCAGATTCCAACAGTTTCACCTCGGAGTGGACAC 142 CTGTGGA VH3GGTGAAACTGTTGGAATCTGGAGGAGGCTTGGTA 143 CAGCC VH4GGAGAGTCTCATAGAACCCCCCGGCTGTACCAAG 144 CCTCCT VH5GGGGGGTTCTATGAGACTCTCCTGTGCAGGTTCT 145 GGATTCA VH6CATGTAGAAATCAGTGAAGGTGAATCCAGAACCT 146 GCACA VH7CCTTCACTGATTTCTACATGAACTGGATTCGCCA 147 GCCTGC VH8GCCACTCAGGTGCCTTCCCTGCAGGCTGGCGAAT 148 CCAGTT VH9AGGGAAGGCACCTGAGTGGCTGGGTTTTATTAGA 149 GACAAA VH10TCTGTTGTGTAACCTTTAGCTTTGTCTCTAATAA 150 AACCCA VH11GCTAAAGGTTACACAACAGAGTACAATCCATCTG 151 TGAAGGGG VH12TCTGGAGATGGTGAACCGCCCCTTCACAGATGGA 152 TTGTAC VH13CGGTTCACCATCTCCAGAGATAATACCCAAAACA 153 TGCT VH14GGGTGTTCATTTGAAGATAGAGCATGTTTTGGGT 154 ATTATC VH15CTATCTTCAAATGAACACCCTAAGAGCTGAGGAC 155 ACTGCC VH16TCTCTTGCACAGTAGTAAGTGGCAGTGTCCTCAG 156 CTCTTA VH17ACTTACTACTGTGCAAGAGAGGGCCACACTGCTG 157 CTCCTTTT VH18CTCCTTGGCCCCAGTAATCAAAAGGAGCAGCAGT 158 GTGGCCC VH19GATTACTGGGGCCAAGGAGTCATGGTCACCGTCT 159 CCTCA VH20 TGAGGAGACGGTGACCATGA160 VHVK5′CHO GCATGTTGACCCTGACGCAAGCTTGCCGCCACCA 161 TGGG VHVK3′SIGGGAGTGGACACCTGTGGAGAGAAAGGC 162 VH12 GCGATAGCTGGACTGAATGGATCCTATAAATCTC163 TG

TABLE 4 Oligonucleotides for synthesis of V_(K) gene SEQ Name SequenceID NO: VK1 TCCACAGGTGTCCACTCCGAC 164 VK2AGACTGGGTCATCTTGATGTCGGAGTGGACACCT 165 GTGGA VK3ATCAAGATGACCCAGTCTCCCTCATTCCTGTCTG 166 CATCTG VK4AGAGTGACTCTGTCTCCCACAGATGCAGACAGGA 167 ATGAGGG VK5TGGGAGACAGAGTCACTCTCAACTGCAAAGCAAG 168 TCAGAA VK6GTTTAAGTATTTGTCAATATTCTGACTTGCTTTG 169 CAGTTG VK7TATTGACAAATACTTAAACTGGTATCAGCAAAAG 170 CTGGGA VK8TCAGGAGTTTGGGAGATTCTCCCAGCTTTTGCTG 171 ATACCA VK9GAATCTCCCAAACTCCTGATATATAATACAAACA 172 ATTTGC VK10CCTTGATGGGATGCCCGTTTGCAAATTGTTTGTA 173 TTATATA VK11AAACGGGCATCCCATCAAGGTTCAGTGGCAGTGG 174 ATCTGG VK12GGTGAGTGTGAAATCAGTACCAGATCCACTGCCA 175 CTGAA VK13TACTGATTTCACACTCACCATCAGCAGCCTGCAG 176 CCTGAA VK14CAGAAATATGTGGCAACATCTTCAGGCTGCAGGC 177 TGCTGAT VK15GATGTTGCCACATATTTCTGCTTGCAGCATATAA 178 GTAGG VK16CCCAGTTCCAAACGTGCGCGGCCTACTTATATGC 179 TGCAAG VK17CCGCGCACGTTTGGAACTGGGACCAAGCTGGAGC 180 TGAAAC VK18AAAGTTTAAATTCTACTCACGTTTCAGCTCCAGC 181 TTGGT VK19GTGAGTAGAATTTAAACTTTGCTTCGTCGACTGG 182 ATCC VK20 GGATCCAGTCGACGAAGC 183VHVK5′CHO GCATGTTGACCCTGACGCAAGCTTGCCGCCACCA 184 TGGG VHVK3′SIGGGAGTGGACACCTGTGGAGAGAAAGGC 185 VK3′CHOGCGATAGCTGGACTGAATGGATCCAGTCGACGAA 186 GC

Chimeric heavy and light chain expression vectors have been constructedconsisting of the rat anti-CD52 variable regions linked to human IgG1[Takahashi et al. (1982) Cell 29: 671] or κ [Heiter et al. (1980) Cell22: 197] constant regions. These composite antibody genes were thentransferred to expression vectors for production of recombinantantibody. The antibody genes are under the control of the humancytomegalovirus immediate early promoter. The heavy chain vectorincludes the dhfr gene and the light chain vector the neo gene forselection in mammalian cells. The DNA sequence was confirmed to becorrect for the V_(H) and V_(K) in the chimaeric expression vectors.

Example 2 Construction of Modified Antibody V_(H) and V_(K) Genes

Modified V_(H) and V_(K) genes were constructed by PCR mutagenesis usingthe rat anti-CD52 variable region cassettes generated in Example 1 astemplates. Table 5 lists the oligonucleotides used in the production ofmodified V_(H)S. The following mutations are identified by the Kabatnumber of the residue with the linear number relating to therespectively identified polypeptide acid sequence in parenthesis. DIVHv1(polypeptide SEQ ID NO: 3; polynucleotide SEQ ID NO: 71) included themutations K3Q (3), M18L (18), 137V (37), P40A (40), A41P (41), A44G(44), P45L (45), L48V (48), T74S (77), Q75K (78), M77T (80), T82bS (87),T89V (95), V107T (115), M108L (116), and used oligonucleotidesVHVK5′CHO, DIVH1, DIVH2, DIVH3, DIVH4, DIVH5, DIVH6, DIVH7, DIVH8,DIVH9, DIVH10, and VH12. DIVHv2 (polypeptide SEQ ID NO: 4;polynucleotide SEQ ID NO: 72) included the mutations K3Q (3), M18L (18),A41P (41), L48I (48), T74S (77), Q75K (78), M77T (80), T82bS (87), T89V(95), V107T (115), M108L (116), and used oligonucleotides VHVK5′CHO,DIVH1, DIVH2, DIVH3, DIVH4, DIVH5A, DIVH6A, DIVH7, DIVH8, DIVH9, DIVH10,and VH12. DIVHv3 (polypeptide SEQ ID NO: 5; polynucleotide SEQ ID NO:73) included the mutations L5Q (5), L20I (20), A23S (23), A41P (41),A44G (44), L48I (48), M77T (80), Y79H (82), M82A (85), T89V (95), V107T(115), M108T (106), and used oligonucleotides VHVK5′CHO, DIVH11, DIVH12,DIVH13, DIVH14, DIVH15, DIVH16, DIVH17, DIVH18, DIVH19, DIVH20, andVH12. DIVHv4 (polypeptide SEQ ID NO: 6; polynucleotide SEQ ID NO: 74)included the mutations K3Q (3), M18L (18), I37V (37), P40A (40), A41P(41), A44G (44), P45L (45), L48V (48), T74A (77), Q75K (78), M77S (80),T82bS (87), T89V (95), V107T (115), M108L (116), and usedoligonucleotides VHVK5′CHO, DIVH1, DIVH2, DIVH3, DIVH4, DIVH5, DIVH6,DIVH7, DIVH8, DIVH9, DIVH10, DIVH21, DIVH22 and VH12. DIVHv5(polypeptide SEQ ID NO: 7; polynucleotide SEQ ID NO: 75) included themutations K3Q (3), M18L (18), A41P (41), T74S (77), Q75K (78), M77T(80), T82bS (87), T89V (95), V107T (115), M108L (116) and usedoligonucleotides VHVK5′CHO, DIVH1, DIVH2, DIVH3, DIVH4, DIVH23, DIVH6A,DIVH7, DIVH8, DIVH9, DIVH10, and VH12.

TABLE 5 Oligonucleotides used in the construction of modified ant-CD52V_(H) _(S) SEQ Name Sequence ID NO: VHVK5′CHOGCATGTTGACCCTGACGCAAGCTTGCCGCCACCA 187 TGGG DIVH1CCACTCCGAGGTGCAACTGTTGGAATCTGG 188 DIVH2 CCAGATTCCAACAGTTGCACCTCGGAGTGG189 DIVH3 AGCCGGGGGGTTCTCTGAGACTCTCCTGTG 190 DIVH4CACAGGAGAGTCTCAGAGAACCCCCCGGCT 191 DIVH5AGGGAAGGGACTTGAGTGGGTGGGTTTTATTAGA 192 G DIVH5ACGGGAAAGCACCTGAGTGGATTGGTTTTATTAGA 193 G DIVH6CCACTCAAGTCCCTTCCCTGGAGCCTGGCGGACC 194 CAGTTCATG DIVH6ACCACTCAGGTGCTTTCCCGGGAGGCTGGCGAATC 195 C DIVH7TCTTCAAATGAACTCCCTAAGAGCTGAGGACACT 196 GCCGTTTACTACTG DIVH8AGGGAGTTCATTTGAAGATAGAGGGTGTTTTTGG 197 AATTATCTCTGG DIVH9TGGGGCCAAGGAACACTGGTCACCGTCTCCTCAG 198 G DIVH10GGAGACTGTGACCAGTGTTCCTTGGCCCCAG 199 DIVH11TCCGAGGTGAAACTGCAGGAATCTGGAGGAGGC 200 DIVH12CCAGATTCCTGCAGTTTCACCTCGGAGTGG 201 DIVH13GGGGGTTCTATGAGAATCTCCTGTTCAGGTTCTG 202 G DIVH14GAACCTGAACAGGAGATTCTCATAGAACCCCCCG 203 G DIVH15CGGGAAAGGACCTGAGTGGATTGGTTTTATTAGA 204 G DIVH16CCAATCCACTCAGGTCCTTTCCCGGGAGGCTGGC 205 G DIVH17GCTAACACCCTAAGAGCTGAGGACACTGCCGTTT 206 ACTACTG DIVH18CTCTTAGGGTGTTAGCTTGAAGATGGAGGGTGTT 207 TTGGG DIVH19TGGGGCCAAGGAACTACCGTCACCGTCTCCTCAG 208 G DIVH20GGAGACGGTGACGGTAGTTCCTTGGCCCCAG 209 DIVH21GATAATGCCAAAAACTCCCTCTATCTTCAAA 210 TGAAC DIVH22ATAGAGGGAGTTTTTGGCATTATCTCTGGAG 211 ATGG DIVH23CGGGAAAGCACCTGAGTGGCTGGGTTTTATT 212 AGAG VH12GCGATAGCTGGACTGAATGGATCCTATAAAT 213 CTCTG

Table 6 lists the oligonucleotides used in the production of modifiedV_(K)S. The following mutations are identified by the Kabat numbers ofthe residues and are the same as the linear numbering of therespectively identified polypeptide sequences. DIVKv1 (polypeptide SEQID NO: 8; polynucleotide SEQ ID NO: 76) included the mutations K3Q,F10S, L21I, N22T, K24R, L40P, E42K, S43A, L46S, T56S, I58V, V83I, F87Y,and used oligonucleotides VHVK5′CHO, DIVK1, DIVK2, DIVK3A, DIVK4A,DIVK5B, DIVK6, DIVK7, DIVK8A, DIVK9, DIVK10, and VK3′CHO. DIVKv2(polypeptide SEQ ID NO: 9; polynucleotide SEQ ID NO: 77) included themutations K3Q, F10S, L21I, N22T, L40P, E42K, S43A, I58V, V83I, F87Y, andused oligonucleotides VHVK5′CHO, DIVK1, DIVK2, DIVK3, DIVK4, DIVK5,DIVK6, DIVK7, DIVK8, DIVK9, DIVK10, and VK3′CHO. DIVKv3 (polypeptide SEQID NO: 10; polynucleotide SEQ ID NO: 78) included the mutations K3Q,F10S, L21I, N22T, K24R, L40P, E42K, S43A, T56S, I58V, V83I, F87Y, andused oligonucleotides VHVK5′CHO, DIVK1, DIVK2, DIVK3A, DIVK4A, DIVK5,DIVK6, DIVK7, DIVK8A, DIVK9, DIVK10, and VK3′CHO. DIVKv4 (polypeptideSEQ ID NO: 11; polynucleotide SEQ ID NO: 79) included the mutations K3Q,F10S, L21I, N22T, L40P, E42K, S43A, L46S, I58V, V83I, F87Y, and usedoligonucleotides VHVK5′CHO, DIVK1, DIVK2, DIVK3, DIVK4, DIVK5B, DIVK6,DIVK7, DIVK8, DIVK9, DIVK10, and VK3′CHO. DIVKv5 (polypeptide SEQ ID NO:12; polynucleotide SEQ ID NO: 80) included the mutations K3Q, F10S,L21I, N22T, L40P, E42K, I58V, V83I, F87Y, and used oligonucleotidesVHVK5′CHO, DIVK1, DIVK2, DIVK3, DIVK4, DIVK5A, DIVK6A, DIVK7, DIVK8,DIVK9, DIVK10, and VK3′CHO.

TABLE 6 Oligonucleotides used in the construction of modified ant-CD52V_(K) _(S) SEQ Name Sequence ID NO: VHVK5′CHOGCATGTTGACCCTGACGCAAGCTTGCCGCCACCA 214 TGGG DIVK1ATGACCCAGTCTCCCTCATCCCTGTCTGCATC 215 DIVK2GAGGGAGACTGGGTCATCTGGATGTCGGAGTGGA 216 C DIVK3CAGAGTCACTATCACCTGCAAAGCAAGTCAGAAT 217 DIVK3ACAGAGTCACTATCACCTGCAGAGCAAGTCAGAAT 218 DIVK4ATTCTGACTTGCTTTGCAGGTGATAGTGACTCTG 219 T DIVK4AATTCTGACTTGCTCTGCAGGTGATAGTGACTCTG 220 T DIVK5CCCGGAAAAGCTCCCAAACTCCTGATATATAATA 221 C DIVK5ACCCGGAAAATCTCCCAAACTCCTGATATATAATA 222 C DIVK5BCCCGGAAAAGCTCCCAAATCCCTGATATATAATA 223 C DIVK6TTTGGGAGCTTTTCCGGGCTTTTGCTGATACC 224 DIVK6ATTTGGGAGATTTTCCGGGCTTTTGCTGATACC 225 DIVK7 CGTCCCATCAAGGTTCAGTGGCAGTGG226 DIVK8 GCCACTGAACCTTGATGGGACGCCCGTTTGC 227 DIVK8ACACTGAACCTTGATGGGACGCCAGATTGCAAATT 228 G DIVK9GCCTGAAGATATTGCCACATATTACTGCTTGCAG 229 C DIVK10TGCAAGCAGTAATATGTGGCAATATCTTCAGGCT 230 G VK3′CHOGCGATAGCTGGACTGAATGGATCCAGTCGACGAA 231 GC

The modified V_(H) and V_(K) expression cassettes produced were clonedas HindIII to BamHI fragments (DNA and amino acid sequences forDIVHYv1-DIVHv5 are shown in FIG. 3-FIG. 7 and for DIVKv1-DIVKv5 areshown in FIG. 8-FIG. 12 respectively) into the plasmid vector pUC 19 andthe entire DNA sequence was confirmed to be correct for each modifiedV_(H) and V_(K).

The modified V_(H) and V_(K) expression cassettes were linked to humanIgG1 (SEQ ID NO: 139; FIG. 13) [Takahashi et al. (1982) Cell 29: 671]and κ (SEQ ID NO: 140; FIG. 14) [Heiter et al. (1980) Cell 22: 197]constant regions respectively. These composite antibody genes were thentransferred to expression vectors for production of recombinantantibody. The antibody genes are under the control of the humancytomegalovirus immediate early promoter. The heavy chain vectorincludes the dhfr gene and the light chain vector the neo gene forselection in mammalian cells. The DNA sequence was confirmed to becorrect for the V_(H) and V_(K) in the expression vectors.

Example 3 Expression, Purification and Quantitation of Anti-CD52Antibodies

The host cell line for antibody expression was CHO dhFr⁻, obtained fromthe European Collection of Animal Cell Cultures, Porton UK (ECACC No94060607). The heavy and light chain expression vectors wereco-transfected into CHO cells by electroporation. Colonies expressingthe neo and dhfr genes were selected in Iscove's Modified Dulbecco'sMedium (IMDM) without nucleosides, supplemented with 10% dialysed foetalbovine serum and 400 μg/ml geneticin (G-418 sulphate) (all from Gibco,Paisley, UK). Transfected cell clones were screened for production ofhuman antibody by ELISA for human IgG [Tempest et al. (1991)BioTechnology 9: 266]. Cell lines secreting antibody were expanded andthe highest producers selected and frozen down in liquid nitrogen. Theanti-CD52 antibodies were purified using Prosep®-A (Bioprocessing Ltd)according to the manufacturer's instructions. The concentration wasdetermined by ELISA for human IgG1 κ antibody.

The assay was conducted in 96-well plates and all determinations wereconducted in duplicate. For the assay, plates (Dynatech Immulon 2) werecoated using 100 μl per well of sheep anti-human κ antibody (The BindingSite, Birmingham, UK) diluted 1:250 in carbonate/bicarbonate coatingbuffer pH9.6 (Sigma, Poole, UK). Coating was conducted for 1 hr at 37°C. and the wells washed 3 times with PBST (PBS with 0.05% Tween 20). Thewells were filled with 100 μL of PBST and the dilutions for the controland test antibodies set out. The negative control uses PBST only and noantibody was added. The standard antibody (Human IgG1/κ purified myelomaprotein, The Binding Site, UK) was diluted to 2 micrograms per ml inPBST. 100 μL was added to duplicate wells in the first column (giving afinal concentration of 1 μg/ml) and doubling dilutions made across theplate. Doubling dilution series were also set out for the test antibodypreparations. The plate was incubated at room temperature for 1 hr andthe wells washed as previously. Bound antibody was detected using aperoxidase conjugated sheep ant-human IgG γ chain specific reagent (TheBinding Site, Birmingham, UK). This secondary antibody was diluted1:1000 in PBST and 100 μl added to each well of the plate. The plate wasincubated for a further 1 hour at room temperature and washed aspreviously. Detection was with o-phenylene diamine (OPD) substrate. Onetablet (20 mg) of OPD (Sigma, Poole, UK) was dissolved in 45 ml ofperoxidase buffer (Sigma, Poole, UK) with 10 μL 30% (w/w) hydrogenperoxide (Sigma, Poole, UK) added just before use. 100 μL of substratewas added per well and incubated at room temperature for five minutes oras required. Color development was stopped by adding 25 μL of 12.5%H₂SO₄ and the results at 492 nm. Antibody concentration versus A₄₉₂ wasplotted and the concentration of the sample antibody determined bycomparison with the standard antibody curve.

Example 4 Testing of Modified Anti-CD52 Antibodies Using a Binding Assay

Human T-cell lymphoma cell line HUT-78 is CD52 positive and was used toassess binding of the modified antibodies of the present invention. Inthe present example, different concentrations of test antibody wereincubated with the cells and the amount of bound antibody was assessedfollowing incubation with a fluorescent-labelled reporter reagent. Thereporter is measured using a fluorescence activated cell sorter (FACS).

Briefly, for each assay, 10⁶ HUT-78 cells were incubated with serialdilutions of test antibody and humanised (CAMPATH-1H) and chimaericanti-CD52 antibodies as controls. The concentrations of the antibodiesin ng/ml were: 40000, 20000, 10000, 5000, 2500, 1250, 625, 312.5,156.25, 78.125, 39.06, 19.53 and 0. All incubations were carried out ina 96 well plate in a final volume of 100 μl PBS/2% FBS.

The antibody and cell mixtures were incubated on ice in the dark for 1hr and washed twice with 200 μl of cold PBS/2% FBS.

For detection, the cells were incubated for 1 hour on ice with a 1:1000dilution of FITC-labelled anti-human IgG Fc domain. This reagent is agoat anti-human IgG (Fc specific) obtained from Sigma (Poole, UK). Thecells were washed as previously and re-suspended in 100 μl of PBS/2% FBSand transferred to 4 ml FACS tubes (Becton Dickinson) containing 900 μlof PBS/2% FBS/Propidium Iodide (1:1000). The cells were analysed using aconventional Becton Dickenson FACS Calibur instrument.

The binding of the test and control antibodies was determined using theMedian Fluorescence value. The saturating concentration of antibody wasdetermined from plots of the Median Fluorescence—Zero Antibody MedianFluorescence versus Concentration of antibody. The binding curves werefitted to a logistic 4 parameter sigmoidal equation using SigmaPlot,giving an excellent fit with 95% confidence levels. The titres, i.e.,concentrations at which 50% of maximum binding occurred, are shown inTable 7. The results indicate that many of the antibodies of the presentinvention show near equivalent binding to the chimeric CAMPATH-1G andthe humanized CAMPATH-1H antibodies.

TABLE 7 Titre (μg/ml) (Concentration which gave 50% Antibody of maximumbinding) Humanised CAMPATH-1H 1.49, 1.44, 2.62, 2.99 ChimaericCAMPATH-1G 1.03, 1.99, 2.55, 2.35, 4.20 DIVH1/DIVK1 2.99 DIVH1/DIVK21.66 DIVH1/DIVK3 1.71 DIVH1/DIVK4 3.45 DIVH1/DIVK5 1.85 DIVH2/DIVK1 5.56DIVH2/DIVK2 3.70 DIVH2/DIVK3 3.89 DIVH2/DIVK4 6.21 DIVH2/DIVK5 1.18DIVH3/DIVK1 9.60 DIVH3/DIVK2 17.79 DIVH3/DIVK3 >40.0 DIVH3/DIVK4 8.63DIVH3/DIVK5 3.30 DIVH4/DIVK1 4.43 DIVH4/DIVK2 1.59 DIVH4/DIVK3 2.28DIVH4/DIVK4 8.54 DIVH4/DIVK5 2.39 DIVH5/DIVK1 4.01 DIVH5/DIVK2 2.45DIVH5/DIVK3 2.55 DIVH5/DIVK4 4.05 DIVH5/DIVK5 3.00

Example 5 Testing of Modified Anti-CD52 Antibodies Using a CompetitionAssay

Competition binding assays were conducted using the modified antibodiesof the present invention. In these assays the test antibodies wereassessed for their ability to compete for binding to CD52 against thehumanised CAMPATH-1H reagent. In the present example, HUT-78 cells areco-incubated with a sub-saturating amount of a biotinylated CAMPATH-1Hand several concentrations of competing non-labelled test antibody. Theamount of biotinylated reference antibody bound to the cells wasdetermined following further incubation with an avidin-FITC reporter andfluorescence determination using a FACS instrument as per Example 4.

Briefly, for each competition assay, 10⁶ HUT-78 cells were incubatedwith 2μg biotinylated human CAMPATH-1H. Pilot experiments had beenpreviously conducted with the biotinylated CAMPATH-1H and unlabelledCAMPATH-1H to determine the optimum amount of biotinylated antibodyrequired for subsequent to addition to each assay.

Serial dilutions of the test and control antibodies were set out into 96well plates in a final volume of 100 μl PBS/2% FBS. Test antibodies wereset out at 0, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, 100, 500, & 1000 μg/10⁶cells.

The cell and antibody mixtures were incubated on ice in the dark for 1hour and washed twice with 200 μl of ice-cold PBS/2% FBS. The boundbiotinylated antibody was detected by incubation with a 1:200 dilutionof an avidin-FITC reagent (Sigma, Poole, UK). Incubation was for 1 houron ice followed by two cycles of washing as previously. The cells werere-suspended in 100 μl of PBS/2% FBS and transferred to 4 ml tubescontaining 900 μl of PBS/2% FBS/Propidium Iodide (diluted 1:1000). Thecells were analysed using a Becton Dickenson FACS Calibur instrument.

The binding of the test and control antibodies was expressed as aper-cent inhibition relative to the maximal binding of the biotinlabelled control.

The percent inhibition value was determined as below:

${\% \mspace{14mu} {Inhibition}} = {\frac{\begin{bmatrix}{{\% \mspace{14mu} {of}\mspace{14mu} {Gated}\mspace{14mu} {Cells}\mspace{14mu} {No}\mspace{14mu} {Competitor}} -} \\{\% \mspace{14mu} {of}\mspace{14mu} {Gated}\mspace{14mu} {Cells}\mspace{14mu} {with}\mspace{14mu} {Competitor}}\end{bmatrix}}{\left\lbrack {\% \mspace{14mu} {of}\mspace{14mu} {Gated}\mspace{14mu} {Cells}\mspace{14mu} {No}\mspace{14mu} {Competitor}} \right\rbrack} \times 100}$

The binding curves were fitted to a logistic 4 parameter sigmoidalequation using SigmaPlot, giving an excellent fit with 95% confidencelevels. The EC₅₀ values were calculated and are shown in Table 8. Theresults indicate that the antibodies of the present invention bind toCD52 on HUT-78 cells with equivalent efficiency to the chimericCAMPATH-1G and the humanized CAMPATH-1H antibodies.

TABLE 8 Antibody EC₅₀ Humanised 1.13, 1.43, 1.00 CAMPATH- 1H Chimaeric1.00, 2.02, 0.87 CAMPATH- 1G DIVH1/DIVK2 2.15, 2.84 DIVH1/DIVK3 0.93,2.20 DIVH1/DIVK5 1.95, 2.75 DIVH2/DIVK5 0.79, 1.04 DIVH4/DIVK2 1.25,2.05 DIVH4/DIVK3 2.19, 2.40 DIVH4/DIVK5 2.20 DIVH5/DIVK1 2.05DIVH5/DIVK2 2.25, 1.65 DIVH5/DIVK3 1.97, 1.10 DIVH5/DIVK5 1.39, 2.43

Example 6 T Cell Immunogenicity Analysis

Modified antibody CAMPATH-1G DIVHv2/DIVKv5, was prepared from the cellline CHO CAMPATH-1G DIVH2/DIVK5 grown in CHO Protein-free AnimalComponent-Free Medium (Sigma Cat No: G7513) supplemented withL-glutamine and Antibiotic-Antimycotic (Gibco/Invitrogen Cat No:15240-062). Antibody was purified by PROSEP-A chromatography(Millipore), eluted with 0.1M glycine pH3.0, neutralised and dialysedagainst phosphate buffered saline (PBS), and finally sterilised byfiltration.

Both the DIVH2/DIVK5 modified antibody and humanised CAMPATH controlwere subjected to a 2-stage purification using cation exchange and sizeexclusion chromatography. After buffer exchange into 50 mM MES pH6 on aSephadex G25 (PD10 column), the protein was passed through a cationexchange column (Mono-S 10/10) and eluted with a sodium chloridegradient (0 to 0.5M). The eluted protein containing fractions were thenapplied to a Superdex 200 preparative column (XK16/60) run in PBS. Peakfractions were pooled and stored at 4° C. The antibody concentrationswere determined by ELISA for human IgG.

Experimental: It was suspected that the anti-CD52 CAMPATH antibody woulditself be inhibitory to T cells, and would interfere with the analysisof immunogenicity in the standard T cell assay. Preliminary experimentswere carried out to test the effect of CAMPATH anti-CD52 antibody on Tcells. PBMC were prepared from blood from three healthy normal donors.These were incubated with humanised CAMPATH-1H (supplied by Ilex) alone,Keyhole Limpet Haemocyanin (KLH) alone, KLH and CAMPATH-1H antibodytogether and untreated control. The results showed that there is acompete inhibition of the response to the control antigen KLH, in all 3donors, due to the effect of the antibody on the T cells.

In order to analyze the immunogenicity of intact anti-CD52 antibody, amore complex T cell assay protocol was used where dendritic cells (DC)were loaded with whole anti-CD52 antibody and exogenous (non-processed)antigen was removed by washing prior to addition of autologous T cells.In this way, the inhibitory effect of anti-CD52 was avoided and normalresponses to KLH achieved. A total of 10 healthy donors were used inthis alternative protocol using humanized CAMPATH-1H as a test controlantigen.

Briefly, PBMC were used as a source of monocytes, which were isolated byadherence to tissue culture plastic (>90% CD14⁺). Monocytes werecultured in AIM V medium (Gibco) with 3% heat inactivated human AB serum(Autogen Bioclear) (growth medium) at an approximate density of 1×10⁶per well (24-well plate). To induce an APC-like phenotype (CD40⁺,CD80^(hi), CD83^(hi), CD86^(hi), MHC class II^(hi)) monocytes wereincubated in growth medium containing human IL-4 (Peprotech) and GM-CSF(Peprotech) for 4 days. On day 4, 50 μg/ml of test antigen (humanisedCAMPATH-1H or modified CAMPATH-1G DIVHv2/DIVKv5 antibody) was added.Control wells received medium only. After 24 hrs the growth medium andantigen was removed and the cells washed once before adding fresh growthmedium containing TNFα (Peprotech), GM-CSF and IL-4 for 4 days. Thenboth adherent and non-adherent dendritic cells (DCs) were harvested andcounted. The DCs were distributed at 1×10⁴ per well of 96 well roundbottom plates, in sextuplicate cultures per treatment (humanisedCAMPATH-1H or modified CAMPATH-1G DIVHv2/DIVKv5 antibody or control) perdonor. The DC were gamma irradiated with 4000 rads before addingautologous CD4⁺ T cells that were negatively isolated from PBMC (DynalHuman CD4⁺ Negative Isolation Kit) at 1×10⁵ per well. Plates wereincubated for 7 days and proliferation was measured by incorporation oftritiated thymidine (a 6-hr pulse with ³H-Thymidine at 1 μCi/well).These data are expressed as a stimulation index where:

${{Stimulation}\mspace{14mu} {Index}} = \frac{{CPM}\mspace{14mu} {of}\mspace{14mu} {test}\mspace{14mu} {antigen}}{{CPM}\mspace{14mu} {of}\mspace{14mu} {untreated}\mspace{14mu} {control}}$

A positive result is defined as a stimulation index (SI) greater than 2.Preliminary results (FIG. 15) show that 2 out of 10 these donorsresponded to CAMPATH-1H, one with a very high stimulation index.

Comparison of CAMPATH-1H and modified DIVHv5/DIVKv2 antibody: A panel oftwenty healthy donors were selected based on HLA-DR typing (see Table 9)for screening the humanised and modified antibodies in T cell assays.This enabled the screening of the antibodies against greater than 80% ofDR alleles expressed in the world population.

TABLE 9 HLA DR haplotypes of the set of 20 healthy donors used to testthe immunogenicity of humanised and modified CAMPATH antibodies DONORAllotype 1 DRB1*04, DRB4*01 2 DRB1*03, DRB1*04, DRB4*01, DRB5 3 DRB1*01,DRB1*13, DRB3 4 DRB1*01, DRB1*07, DRB4*01 5 DRB1*11 AND DRB1*13 OR 14,DRB3 6 DRB1*03 AND DRB1*08, 11 OR 13, DRB3 7 DRB1*01, DRB1*11, DRB3 8DRB1*10, DRB1*15, DRB5 9 DRB1*04, DRb1*15, DRB4*01, DRB5 10 DRB1*03,DRB1*15, DRB3, DRB5 11 DRB1*13, DRB1*16, DRB3, DRB5 12 DRB1*03, DRB1*07,DRB3, DRB4 13 DRB1*03, DRB1*10, DRB3 14 DRB1*04, DRB1*09, DRB4*01 15DRB1*09, DRB1*15, DRB4*01, DRB5 16 DRB1*03, DRB1*08, DRB3 17 DRB1*08,DRB1*15, DRB5 18 DRB1*13&DRB1*14 OR DRB13, DRB3 19 DRB1*07, DRB4*01 20DRB1*07, DRB1*16, DRB4*01, DRB5

FIG. 16 shows that humanised CAMPATH 1H induced significant (p<0.05)proliferative responses (cpm compared to untreated controls) in threehealthy individuals (donors 14, 17 and 19). However only T cells fromdonors 14 and 17 produced sufficiently high (SI>2) stimulation indexesof 4.2 and 2.5, respectively. The donor 19 response was excluded sincethe stimulation index was considerably lower (SI˜1.5) than the thresholdset for this experiment. For Donor 8 the untreated control produced lessthan 400 cpm and was therefore excluded from the study. Importantly,none of the donors responded to the modified DIVHv5/DIVKv2 antibody.

Thus, the humanised CAMPATH 1H antibody has the potential to induce a Tcell dependent humoral immune response (marked by affinity matured,isotype switched anti-CAMPATH 1H antibodies) in some human patients withcertain MHC Class II allotypes. This observation was supported by exvivo T cell assays in which T cell activation occurred in at least twohealthy individuals (donors 14 and 17) in response to treatment withantigen processed CAMPATH 1H (expressed by matured DC). Comparison of exvivo T cell responses using antigen processed modified DIVHv5/DIVKv2antibody showed that this completely failed to induce T cellproliferation in any of the donors tested. These data demonstrate thatthe modified antibody is likely to provide an improved therapeuticmolecule when substituted for humanised CAMPATH-1H, particularly whenused for indications where repeated dosing is required.

1-22. (canceled)
 23. An expression vector comprising: a) a first nucleicacid sequence coding for a V-region heavy chain comprising a substitutedvariant of SEQ ID NO: 1 with one or more of the following substitutionswherein the numbering of amino acid residues relates to those of SEQ IDNO: 1: substitution of Lys at amino acid residue 3 with Gln; Leu atamino acid residue 5 with Ala, Cys, Asn, Asp, Gln, Glu, Gly, His, Lys,Pro, Arg, Ser, or Thr; Val at amino acid residue 12 with Asn, Asp, Glu,His, Lys, Pro, Gln, Arg, Ser, or Thr; Gln at amino acid residue 13 withAla, Phe, His, Lys, Asn, Pro, Gln, Arg, Ser, Thr; Gly at amino acidresidue 15 with Asp, His, Pro, Gln, Arg, Ser, Thr; Ser at amino acidresidue 17 with Gly, Met, Pro or Trp; Met at amino acid residue 18 withArg, Gly, Pro, Leu; Arg at amino acid residue 19 with Ala, Cys, Phe,Gly, Ile, Leu, Met, Pro, Val, Trp, or Tyr; Leu at amino acid residue 20with Ala, Cys, Phe, Gly, His, Ile, Lys, Asn, Asp, Met, Gln, Glu, Pro,Arg, Ser, Thr, Val Trp, or Tyr; Ser at amino acid residue 21 with Pro;Ala at amino acid residue 23 with Asn, Asp, Glu, Gln, Gly, His, Lys,Pro, Arg, Ser, or Thr; Ser at amino acid residue 25 with Phe, Gly, Leu,Pro, Trp or Tyr; Gly at amino acid residue 26 with Asp, Asn, Glu, Gln,His, Lys, Pro, Arg, Ser, Thr, Trp or Tyr; Asp at amino acid residue 31with Ala, Phe, Gly, Ile, Met, Pro, Val, Trp, or Tyr; Tyr at amino acidresidue 33 with Ala, Gly, Met, or Pro; Asn at amino acid residue 35 withPro; Trp at amino acid residue 36 with Ala, Asp, Glu, Gly, His, Lys,Asn, Pro, Gln, Arg, Ser, or The; Ile at amino acid residue 37 with Val;Arg at amino acid residue 38 with Phe, His, Pro, or Tyr; Pro at aminoacid residue 40 with Ala; Ala at amino acid residue 41 with Asp, Asn,Glu, Gln, His, Lys, Pro, Arg, Ser, The or Trp; Gly at amino acid residue42 with Ile, Pro, Thr, Tyr; Ala at amino acid residue 44 with Gly, His,Asn, Pro, Gln, Ser, Thr, Trp, Tyr, Pro at amino acid residue 45 withLeu; Leu at amino acid residue 48 with Val or Ile, Thr at amino acidresidue 71 with Phe, Leu, Pro, Trp, or Tyr; Ile at amino acid residue 72with Asp, Glu, His, Lys, Asn, Pro, Gln, Arg, Ser, or Thr; Ser at aminoacid residue 73 with Ala, Gly, or Pro; Arg at amino acid residue 74 withAla, Phe, Gly, Ile, Met, Pro, Trp, or Tyr; Thr at amino acid residue 77with Ala, His, Ile, Pro or Ser; Gln at amino acid residue 78 with Lys;Asn at amino acid residue 79 with Ala, Phe, Gly, Ile, Met, Pro, Val, Trpor Tyr; Met at amino acid residue 80 with Ala, Asp, Glu, Gly, His, Lys,Asn, Pro, Gln, Arg, Thr, or Ser; Tyr at amino acid residue 82 with Ala,Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg, Ser or Thr; Gln at aminoacid residue 84 with Ala, Phe, Gly, Ile, Leu, Met, Pro, Val, Trp or Tyr;Met at amino acid residue 85 with Ala, Asp, Glu, Gly, His, Lys, Asn,Pro, Gln, Arg, Ser or Thr; Thr at amino acid residue 87 with Ser; Leu atamino acid residue 88 with Asp, Glu, Gly, His, Lys, Asn, Pro, Gln, Arg,Ser, or Thr; Arg at amino acid residue 89 with Phe, Pro, Trp, Tyr; Alaat amino acid residue 90 with Asn, Asp, Glu, Gln, His, Lys, Pro, Arg,Ser, Thr, Trp or Tyr; Glu at amino acid residue 91 with Pro; Asp atamino acid residue 92 with Ala, Phe, Gly, Ile, Leu, Met, Pro, Val, Trp,Tyr; Thr at amino acid residue 95 with Val; Asp at amino acid residue109 with Ala, Phe, Gly, Ile, Leu, Met, Pro, Val, Trp or Tyr; Trp atamino acid residue 111 with Ala, Asp, Glu, Gly, His, Lys, Asn, Pro, Gln,Arg, Ser, or Thr; Gly at amino acid residue 114 with His, Pro, Ser, orThr; Val at amino acid residue 115 with Thr; Met at amino acid residue116 with Thr, Phe, Ile, Leu, Pro, Val, Trp or Tyr; Val at amino acidresidue 117 with Ala, Phe, Gly, Ile, Met, Pro, Trp or Tyr; and b) asecond nucleic acid sequence coding for a V-region light chaincomprising a substituted variant of SEQ ID NO:2 with one or more of thefollowing substitutions wherein the numbering of amino acid residuesrelates to those of SEQ ID NO: 2: substitution of Lys at amino acidresidue 3 with Gln; Phe at amino acid residue 10 with Ala, Asp, Asn,Glu, Gln, Gly, His, Lys, Pro, Arg, Ser or Thr; Val at amino acid residue15 with Ala, Gly, His or Pro; Asp at amino acid residue 17 with Pro; Valat amino acid residue 19 with Pro or Trp; Leu at amino acid residue 21with Pro or Ile; Asn at amino acid residue 22 with Thr; Lys at aminoacid residue 24 with Arg; Leu at amino acid residue 33 with Ala, Asp,Asn, Gln, Glu, Gly, His, Lys, Pro, Arg, Ser or Thr; Leu at amino acidresidue 40 with Asp, Asn, Gln, Glu, Gly, His, Lys, Pro, Arg, Ser or Thr;Glu at amino acid residue 42 with Lys; Ser at amino acid residue 43 withAla; Leu at amino acid residue 46 with Ser; Thr at amino acid residue 56with Ala, Phe, Gly, Ile, Met, Pro, Ser, Trp or Tyr; Ile at amino acidresidue 58 with Ala; Gly, Met, Pro or Val; Ser at amino acid residue 60with Ala, Phe; Gly, Ile, Met, Pro; Trp or Tyr; Arg at amino acid residue61 with Pro; Ser at amino acid residue 63 with Phe, Leu, Pro, Trp orTyr; Gly at amino acid residue 64 with Asp, Asn, Gln, Glu, His, Lys,Pro, Arg, Ser or Thr; Leu at amino acid residue 78 with Asp, Asn, Gln,Glu, Gly, His, Lys, Pro, Arg, Ser, Thr; Val at amino acid residue 83with Ala, Asp, Asn, Glu, Gln, Gly, His, Ile, Lys, Pro, Arg, Ser, Thr;Phe at amino acid residue 87 with Tyr, wherein the first nucleic acidsequence and the second nucleic acid sequence are operably linked to oneor more expression control sequences.
 24. An expression vectorcomprising a nucleic acid sequence coding for SEQ ID NO: 71, SEQ ID NO:73, SEQ ID NO: 74 or SEQ ID NO: 75, or a degenerate variant thereof. 25.(canceled)
 26. The expression vector of claim 23, further comprising anucleic acid sequence coding for a human IgG1 constant region domain.27. A cultured cell comprising the vector of claim
 23. 28. (canceled)29. An expression vector comprising a nucleic acid sequence coding forSEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79, or adegenerate variant thereof.
 30. (canceled)
 31. The expression vector ofclaim 23, further comprising a nucleic acid sequence coding for a humankappa constant region domain. 32-33. (canceled)
 34. A method ofpreparing an immunoglobulin, comprising culturing the cell of claim 27under conditions permitting expression under the control of theexpression control sequences, and purifying the immunoglobulin from themedium of said cell, wherein the immunoglobulin has specificity for CD52antigen.